Use of sterols as active ingredient in a cosmetic composition against adiposity

ABSTRACT

The present invention relates to the use of sterols derived from a plant or algae extract as the active principle of a cosmetic composition for combating adiposity. Said sterols are, for example, at least one of the three following sterols: campesterol, beta-sitosterol or stigmasterol.

[0001] The present invention relates to a use of sterols as activeprinciple in a cosmetic composition to combat adiposity and thus refinethe silhouette.

[0002] Sterols are known for their use as active substance inanti-inflammatory medications in which they act as inhibitors oflipoxygenase. One may refer to the patent document FR-A-2 705 030 whichshows such an activity.

[0003] They are also known for cosmetic compositions for treatment ofthe skin in which they act as emollients by presenting good propertiesof moisturizing and barrier. Document U.S. Pat. No. 4,604,281 shows sucha use.

[0004] Concerning cosmetic compositions to combat adiposity, one isacquainted with those of which the active principles used present alipolytic activity by which they stimulate the degradation (or lysis) ofthe fats stored in the human adipocytes, in particular in the form oftriglycerides, and promote the elimination of the products of thatdegradation, in particular fatty acids and glycerols. For example,theophylline and isoproterenol are known to stimulate the lipolysis oftriglycerides, in particular triglycerides of human adipocytes.

[0005] Moreover, in the French patent document No. 98 02152, the activeprinciple used to stimulate lypolysis is a pyrone group which has theeffect of activating the phosphorylation of a lipase enzyme for thedegradation of the triglycerides in fatty acids. This pyrone group is,for example, mangosteen extracted from a tree of the Garcinianaicitgostana species.

[0006] One is also acquainted with cosmetic compositions the activeprinciple of which is not to stimulate the lipolysis of triglycerides ofhuman adipocytes but to inhibit the lipogenesis of these triglycerides.

[0007] It is to be recalled here that, in the adipocytes, lipolysis isin competition with lipogenesis which consists in the formation of fats,in particular triglycerides, and which brings into play an entire seriesof biochemical reactions bringing about the action of proteins withenzymatic activity such as Acyl-CoA synthetase and fatty acidsynthetase, which are also known under the name FAS (fatty acidsynthase).

[0008] Cerulenine is a compound which is known to inhibit lipogenesis byinhibition of the FAS synthetase.

[0009] In a previous patent application, it was also shown that the sameis true with eicosapentanoic acid (EPA).

[0010] It has now been discovered that sterols which come from a plantor algae extract have, on the one hand, an inhibitory activity forlipogenesis of triglycerides produced by human adipocytes and, on theother hand, an activity stimulating the lipolysis of the samesubstances.

[0011] The invention therefore has as object the use of sterols from aplant or algae extract as active substance in a cosmetic composition tocombat adiposity.

[0012] It is thought that the sterols that are contained in the saidextract are responsible for an inhibitory effect on the action of anenzyme that plays a part in lipogenesis, in particular the synthesis offatty acids by fatty acid synthetase FAS. Thus, by using a cosmeticcomposition that contains sterols as active principle, the lipogenesisof these fatty acids is inhibited or at the very least limited and theirconcentration in the adipocytes is thus reduced.

[0013] We were able to show below that most particularly campesterol andbeta-sitosterol, generally present in plants or algae, have thisinhibitory action.

[0014] It is to be understood that the cosmetic compositions that areplanned to inhibit this lipogenesis make it possible to avoid theinstantaneous recovery of the fat lost after lipolysis.

[0015] It is also thought that the sterols have a direct effect on thelipolysis of the triglycerides of human adipocytes by stimulating it.

[0016] We were able to show below that this is particularly trueconcerning the stigmasterol derived from a plant or algae extract.

[0017] It is to be understood that the cosmetic compositions that areplanned to stimulate lipolysis have a direct curative effect by reducingexcess fat.

[0018] It is also thought that sterols have an inhibitory effect on thedifferentiation of adipocytes. In fact, it has been demonstrated thatsteroid hormones (estrogens and androgens) regulate the differentiationof adipocytes, that is to say the maturation of dermic cells(fibroblasts) into cells capable of accumulating fat in the form oftriglycerides.

[0019] Moreover, androgens such as testosterone, dihydrotestosterone(DHT), dehydroepiandrosterone (DHEA), androstanediol and androstenediolhave an inhibitory effect on the differentiation of adipocytes, inparticular by the inhibition of the activity of the enzyme glucose-6phosphate dehydrogenase, while the action of the estrogens likebeta-estradiol is expressed by a modulation of the expression ofreceptors like that of IGF1 (insulin growth factor 1).

[0020] During differentiation, the size of the cells and the fat contentincrease greatly, and “markers” also appear, such as, for example, anincrease in expression of enzymes like lipoprotein lipase (LPL), adecrease in enzymatic activity like that of glycerol 3-phosphatedehydrogenase, an increase in the expression of membrane proteins likethe fatty acid transporter (FAT), an increase in the expression oftranscription factors of the genes of lipogenesis like the peroxisomeproliferator-activated receptors (PPARs) or the CCAAT/enhancer-bindingproteins (C/EBPs), and an increase in the quantity of intracellularproteins like p18 (INK4c), p21 (Waf1) or aP2 (adipocyte fatty acidbinding protein or FABP).

[0021] It may therefore be thought that the effect of phytosterols isexpressed by an effect on the differentiation of pre-adipocytes intoadipocytes (anti-adipogenic effect), in addition to the inhibitoryeffect on lipogenesis and the lipolytic effect.

[0022] The cosmetic compositions that are planned for inhibiting thedifferentiation of adipocytes will have a preventive effect bypreventing the development of excesses of fat.

[0023] According to the present invention, the extract containing atleast one sterol is an oily extract obtained from seeds of Polygonumfagopyrum.

[0024] The present invention also relates to a preparation process of asubstance that contains as active principle a lipidic extract of plantsor algae containing sterols.

[0025] We will now describe the procedure of the experiments that wereconducted to prove the effect of sterols derived from an extract ofplants or algae on lipids.

[0026] In these experiments, adipocytes were isolated from abdominalrestorative surgeries sampled in women. The experiment took place theday of the sampling. Fragments of adipose tissues were incubated for 30minutes at 37° C. in the presence of collagenase, then the isolatedadipocytes were washed and taken up in medium MEM buffered withbicarbonate, in the presence of antibiotics (penicillin/streptomycin),glutamine (2 mM), and delipidated bovine serum albumin (0.5%).

[0027] The tests concerning the effect on lipogenesis were thenconducted in the following manner.

[0028] Adipocytes (90 μl of ¼ diluted suspension) were incubated for 1hour, either with a control medium (4 hours), with molecules known toinhibit the neosynthesis of fatty acids (EPA), as for example insulin,or with previously diluted extracts (added under 100 μi).

[0029] As to the sterols and to the lipidic extracts of plants or algae,they were diluted in mineral oil which has been verified to not inhibitincorporation or extraction of adipocyte lipids. Then, 10 μl of[2¹⁴C]-acetate, 50 μCi/ml, were added, and the samples were eithercultivated at 37° C. in the presence of 5% CO₂ for 4 additional hours,or immediately frozen at −80° C. (control to).

[0030] Each experiment was carried out in duplicate insofar as the testproved to be reproducible and with low standard deviations.

[0031] At the times indicated, the samples were frozen at −80° C. Thelipids were then extracted according to the method of Bligh and Dyer(extraction methanol/chloroform/water), then dried under nitrogen. Theradioactivity incorporated was then counted by liquid scintillation.

[0032] As to the tests concerning the effect on lipolysis, they wereconducted in the following manner. Adipocytes (540 μl of suspension)were incubated for 2 hours in a water bath at 37° C., with agitation, inthe presence of 60 μl of different sterols. The experiments were carriedout in triplicate.

[0033] Then, the non-esterified fatty acids (AGNE) released by theadipocytes were determined in the sub-adipocyte media. The valuesobtained were treated by an analysis of variance (ANOVA) by means of themultiple comparison test of Dunnett. The values were considered assignificant when the probability p was less than the threshold ofsignificance 0.05.

[0034] The experimental results are recorded hereinafter.

[0035] Concerning the lipogenesis test, the acetate incorporation after4 hours of labeling was great (control in Table 1 below, namely 208546cpm in the absence of insulin) and the background at t0 was low: 6465cpm, namely 3% of the 4h time.

[0036] It is to be noted that the addition of insulin effectivelyincreases the incorporation of acetate in human adipocytes (see the sameresults as in Table 1 but in the presence of insulin). As a matter offact it is to be noted that the incorporation of acetate increased 16%in the presence of 1 nM of insulin (Table 2).

[0037] With another donor, the increase was 120% in the presence of 100nM of insulin (result not shown).

[0038] It is to be noted that EPA (or eicosapentanoic acid) (C20:5) inoily solution (mineral oil) at the two concentrations tested (0.1 mM and1 mM) greatly inhibited incorporation (82 and 97% inhibitionrespectively). It will be noted that in the presence of insulin (1 nM),the effect of the EPA at 0.1 mM is slightly decreased (62% instead of82%) but it was still 97% at 1 mM EPA.

[0039] Three sterols were tested in the model described above:campesterol, beta-sitosterol, and stigmasterol. They were diluted inmineral oil the presence of which in the medium has been verified to notinhibit incorporation or extraction of the adipocyte lipids. As for EPAabove, they were tested both in the absence and in the presence ofinsulin (1 nM).

[0040] In the first case, campesterol at 0.01, 0.1 and 1 mM inhibits ina dose-dependent way the incorporation of acetate with respect to themineral oil control (Table 1). At the lowest concentration tested (10μM), the inhibition is 8%. At 0.1 mM, the inhibition is 10% and, at 1mM, it is 26%.

[0041] With beta-sitosterol at 1 mM, the inhibition of acetateincorporation is 16%. At 0.01 and 0.1 mM, this inhibition is weak andnon-significant.

[0042] As to stigmasterol, at the three concentrations tested (0.01, 0.1and 1 mM), the inhibition is weak and non-significant.

[0043] In the second case, in the presence of insulin (1 nM),campesterol at 0.01 mM had no significant effect. By contrast, atconcentrations of 0.1 and 1 mM, it inhibits in a dose-dependent way theincorporation of acetate with respect to the mineral oil control (Table2): the inhibition is 15% at 0.1 nM and 52% at 1 mM. Its inhibitoryeffect is therefore amplified by the presence of insulin.

[0044] It is therefore to be noted that campesterol regulates thesynthesis of triglycerides induced by insulin.

[0045] Beta-sitosterol at 1 mM inhibits the incorporation of acetate by17%. At 0.01 and 0.1 mM, it has no significant effect (inhibition lessthan 10%).

[0046] On the other hand, stigmasterol, at the three concentrationstested (0.01, 0.1 and 1 mM) had no significant effect (inhibition lessthan 5%).

[0047] We now study the same effects as previously but with two oilyextracts obtained from seeds of Polygonum fagopyrum (sarrazin,buckwheat). One of these extracts, designated POL, is obtained byextraction with supercritical CO₂. It contains sterols (approximately3.6%), in particular beta-sitosterol (78% of the sterols), campesterol(7%) and stigmasterol (2%). It was tested at 0.05, 0.2 and 0.5% (dilutedin mineral oil).

[0048] As to the second extract which is a wax designated P, it isobtained by mixture of an extract of co-extraction with supercriticalCO₂ in the presence of a vegetable oil (C8-C10TG) and of a secondvegetable oil solid at ambient temperature, waxy oil, (C16-C18 TG). Thewax obtained presents the advantage of being stable to oxidation(evaluation according to the Rancimat method). It was tested at 0.1, 0.5and 2%.

[0049] Moreover, an excipient, designated MICRE, composed of theextraction solvent C8-C10 TG and the waxy oil C16-C18 TG, was tested.

[0050] As previously, these tests were done at first in the absence ofinsulin (see the results Table 1) and then in the presence of insulin(see the results Table 2).

[0051] In the absence of insulin, it can be noted that the POL extractgreatly inhibits the incorporation of acetate: 74% inhibition at theconcentration of 0.05%, and 91-92% inhibition at the concentrations of0.2 and 0.5% (Table 1).

[0052] The P wax, tested at 0.1%, inhibits the incorporation of acetateby 18%. At 0.5%, it inhibits it by 33%. At the highest concentrationtested (2%), it inhibits it by 68%. It is to be noted that at thatconcentration, the MICRE wax (without the sarrazin extract) inhibited itby 44%.

[0053] In the presence of insulin (1 nM), the POL extract greatlyinhibited the incorporation of acetate: 49, 89 and 91% inhibitionrespectively at the concentrations of 0.05, 0.2 and 0.5% (Table 2).

[0054] The P wax, tested at 0.1%, had no significant effect (5%inhibition). At 0.5%, it inhibited the incorporation of acetate by 42%.At the highest concentration tested (2%), it inhibited it by 57%. Atthat concentration, the MICRE wax (without the sarrazin extract)inhibited it only by 20%.

[0055] We now discuss the tests on lipolysis.

[0056] It can be noted (see Table 3) that basal lipolysis was elevated,but cellular lysis at the end of the experiment was moderate. Thereference molecules (theophylline at 1 nM and isoproterenol at 1 μM)stimulated the lipolysis (p<0.05).

[0057] Campesterol and beta-sitosterol, tested at 10⁻⁶ M, 10⁻⁵ M, 10⁻⁴M, 10⁻³ M and 10⁻² M, did not stimulate lipolysis under the experimentalconditions of this test (Table 3).

[0058] Stigmasterol did not show significant effect at 10⁻⁶ M and at10⁻² M, but it stimulated in a significant way (p<0.05) the liberationof NEFA (non-esterified fatty acids) (26% to 33% increase) at 10⁻⁵ M,10⁻⁴ M and 10⁻³ M. This sterol therefore showed a lipolytic effect underthe experimental conditions.

[0059] The conclusions to be drawn from these experiments are thefollowing.

[0060] Campesterol and beta-sitosterol inhibit the incorporation ofacetate by isolated human adipocytes. Their effect limits lipogenesis.

[0061] Stigmasterol increases the liberation of fatty acids by isolatedhuman adipocytes. Its effect stimulates lipolysis.

[0062] The oily extract of seeds of Polygonum fagopyrum (POL),containing campesterol, beta-sitosterol and stigmasterol, decreases theincorporation of acetate by isolated human adipocytes.

[0063] The buckwheat wax (P wax), containing the extract of Polygonumfagopyrum, also decreases the incorporation of acetate, with an activitygreater than that of the wax alone (without the POL extract). TABLE 1Effect of EPA, 3 sterols and oily extracts of sarrazin on theincorporation of acetate (LIPOGENESIS) in the absence of insulin Averagecpm*- Treatment of cpm t0 test % control % inhibition t0 test 6465 0 0 /Control 208546 202081 100 / EPA 1 mM 1287 6373 3 97 0.1 mM 43253 3678818 82 Campesterol 10⁻³ M 156448 149983 74 26 10⁻⁴ M 188278 181813 90 1010⁻⁵ M 191817 185352 92 8 Beta-sitosterol 10⁻³ M 175711 169246 84 1610⁻⁴ M 198694 192229 95 5 10⁻⁵ M 200044 193579 96 4 Stigmasterol 10⁻³ M210055 203590 101 0 10⁻⁴ M 200467 194002 96 4 10⁻⁵ M 198442 191977 95 5POL 0.5% 24665 18200 9 91 0.2% 23153 16688 8 92 0.05% 58489 52024 26 74P Wax 2% 71676 65211 32 68 0.5% 141501 135036 67 33 0.1% 172219 16575482 18 MICRE 2% 120314 113849 56 44

[0064] TABLE 2 Effect of EPA, 3 sterols and oily extracts of sarrazin onthe incorporation of acetate (LIPOGENESIS) in the presence of insulin (1nM) Average cpm*- Treatment of cpm* t0 test % control % inhibition t0test 6465 0 0 / Control 241167 234702 100 / EPA 1 mM 13384 6919 3 97 0.1mM 96244 89779 38 62 Campesterol 10⁻³ M 119960 113495 48 52 10⁻⁴ M206655 200190 85 15 10⁻⁵ M 243051 236586 101 0 Beta-sitosterol 10⁻³ M201168 194703 83 17 10⁻⁴ M 224445 217980 93 7 10⁻⁵ M 216417 209952 89 11Stigmasterol 10⁻³ M 227385 220920 94 6 10⁻⁴ M 234524 228059 97 3 10⁻⁵ M235529 229064 98 2 POL 0.5% 27264 20799 9 91 0.2% 32221 25756 11 890.05% 125449 118984 51 49 P Wax 2% 106660 100195 43 57 0.5% 143077136612 58 42 0.1% 228281 221816 95 5 MICRE 2% 194695 188230 80 20

[0065] TABLE 3 Effects of 3 sterols on the liberation of NEFA(non-esterified fatty acids) (LIPOLYSIS) Average of the Standard %Treatment NEFA (μM) Deviation Control p Untreated 141 12 100 —Theophylline  1 mM 551 10 390 P < 0.01 Isoproterenol  1 μM 483 28 342 P< 0.01 Campesterol 10⁻² M 167 14 118 p > 0.05 10⁻³ M 138 8 98 p > 0.0510⁻⁴ M 155 6 109 p > 0.05 10⁻⁵ M 140 14 99 p > 0.05 10⁻⁶ M 166 15 117p > 0.05 Beta-sitosterol 10⁻² M 131 17 92 p > 0.05 10⁻³ M 145 11 103 p >0.05 10⁻⁴ M 135 19 96 p > 0.05 10⁻⁵ M 151 2 107 p > 0.05 10⁻⁶ M 156 13110 p > 0.05 Stigmasterol 10⁻² M 158 3 112 p > 0.05 10⁻³ M 178 10 126 p< 0.05 10⁻⁴ M 189 14 133 p < 0.01 10⁻⁵ M 180 21 128 p < 0.05 10⁻⁶ M 15815 112 p > 0.05

1. Method for combating adiposity comprising administering a cosmeticcomposition containing sterols derived from a plant or algae extract asactive principle of the cosmetic composition.
 2. Method according toclaim 1, wherein said sterols are at least one of the three followingsterols: campesterol, beta-sitosterol or stigmasterol.
 3. Methodaccording to claim 2, wherein said sterols are at least one of the twofollowing sterols: campesterol or beta-sitosterol, said cosmeticcompostion being planned to combat adiposity by inhibition oflipogenesis by adipocytes.
 4. Method according to claim 2, wherein saidsterols are at least stigmasterol, said cosmetic composition beingplanned to combat adiposity by stimulation of lipolysis by adipocytes.5. Method according to claim 2, wherein said cosmetic composition isplanned to combat adiposity by inhibition of differentiation ofpre-adipocytes into mature adipocytes.
 6. Method according to claim 1,wherein said extract is an oily extract obtained from seeds of Polygonumfagopyrum.
 7. Method according to claim 2, wherein said extract is anoily extract obtained from seeds of Polygonum fagopyrum.